Biopharma looks to deliver on COVID-19 vaccines and other promising therapies.

Biopharma Gets Back to “New Normal” Business

Can lessons learned during the pandemic pay off in shorter development timelines?

Boosting Drug Development

J&J rollouts out initial vaccine supply; Industry expedites vaccine production

Merck to Produce J&J Vaccine

on March 2, 2021 that, in collaboration with Health Canada, it has published the full clinical data set that was reviewed as part of the authorization of the Moderna COVID-19 vaccine. Publication of data is part of EMA’s efforts to be transparent in its authorization of COVID-19 vaccines.

“With the publication of the data package for Moderna’s COVID-19 vaccine, we are publishing the clinical reports that we assessed in support of the authorisation of this vaccine, and going forward, we will continue with this open data approach for all other COVID-19 vaccines,” said Emer Cooke, EMA’s executive director, in a press release. “EMA and Health Canada’s joint commitment to openness and transparency will support global research, allow for public scrutiny and reinforce society’s trust in COVID-19 vaccines as mass vaccination campaigns continue to be rolled out across the EU, Canada and the rest of the world.”

The published data are available on EMA’s clinical data website and Health Canada’s Public Release of Clinical Information portal. EMA stated that clinical data for the Pfizer-BioNTech vaccine will be published soon.

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The European Medicines Agency and Health Canada have published clinical data used to support the authorization of Moderna’s COVID-19 vaccine.

Clinical Data on Moderna COVID-19 Vaccine Published

A united effort between federal agencies is underway to combat COVID-19 fraud that ranges from the sale of misbranded drugs claiming to treat COVID-19 to products claiming to prevent the virus. The situation is being monitored by FDA’s COVID-19 Fraudulent Products Task Force. Operation Quack Hack has resulted in hundreds of these products being removed from the marketplace, and FDA has sent more than 120 warning letters to various companies selling these fraudulent products. Fake vaccines have also been reported for sale.

“Consumers should know the COVID-19 vaccines that the FDA has authorized under Emergency Use Authorization cannot be sold online. Legitimate COVID-19 vaccines are being distributed for free. Unfortunately, the agency is aware of reports of unauthorized websites and listings on online marketplaces purportedly offering COVID-19 vaccines for sale. When such online listings are found by or reported to the FDA, the agency notifies the online marketplaces, who can then remove the listings. We are also aware of emails sent to consumers featuring the FDA logo, advising consumers to call a phone number to schedule a vaccination. If consumers are contacted directly by someone who says they are from the FDA about a COVID-19 vaccine appointment, it is a scam,” said Judy McMeekin, Pharm. D., associate commissioner for Regulatory Affairs, in an 

FDA is asking the public to help by being alert about COVID-19 fraud schemes. Resources to help consumers protect themselves from fraud can be found on the 

The agency is working to identify and obstruct scammers looking to profit from the pandemic.

FDA Protects Consumers Against COVID-19 Scams

on March 4, 2021 that its human medicines committee (CHMP) has begun a rolling review of the Sputnik V (Gam-COVID-Vac) COVID-19 vaccine. The review is based on results from laboratory and clinical trial studies that indicate the vaccine triggers the production of antibodies and immune cells. The rolling review will look at data as they become available and will continue until there is enough evidence to support a formal marketing authorization application. The usual standards for effectiveness, safety, and quality will be assessed by the agency for the vaccine, according to EMA.

Sputnik V (Gam-COVID-Vac) was developed by Russia’s Gamaleya National Centre of Epidemiology and Microbiology. EMA states that the vaccine is made up of Ad26 and Ad5 adenoviruses that “have been modified to contain the gene for making the SARS-CoV-2 spike protein; they cannot reproduce in the body and do not cause disease. The two adenoviruses are given separately: Ad26 is used in the first dose and Ad5 is used in the second to boost the vaccine’s effect.”

The European Medicines Agency has begun a rolling review based on laboratory and clinical studies of the Sputnik V (Gam-COVID-Vac).

Rolling Review of Sputnik V COVID-19 Vaccine Begins in Europe

Many COVID-19 treatments and vaccines use glass vials and stoppers as their container closure system (CCS), and supply of these components may become constrained, according to FDA. Because of this, manufacturers may need to update approved applications to make changes to CCS components. FDA, therefore, 

 on March 4, 2021 providing recommendations for reporting and implementing changes to COVID-19 CCS components that consist of glass vials and stoppers for parenteral sterile drug products and biologics.

“In response to this public health emergency, FDA will consider risk-based approaches to facilitate implementation of chemistry, manufacturing, and controls (CMC) changes to a CCS in an effort to mitigate the disruptive effect of the COVID-19 pandemic. These measures may include, but may not be limited to, recommendations for adjustments to submission content and reduction in reporting categories for CMC changes,” the guidance states.

The guidance discusses regulatory approaches to CMC changes and common changes to glass vials and stoppers. The guidance also discusses risk-based tools for implementing CCS changes and comparability protocols.

The guidance document provides recommendations for reporting and implementing changes to container closure system components consisting of glass vials and stoppers for sterile drug products.

FDA Publishes Guidance on Changes to COVID-19 Container Closure Systems

The European Commission (EC) has launched a structured dialogue aimed at gaining a better understanding of global medicines supply chains and to identify potential causes and drivers of vulnerabilities.

Bringing together actors of the pharmaceuticals manufacturing chain, public authorities, research communities, health professionals, and patient organizations, the dialogue is one of the flagship initiatives of the EC’s Pharmaceutical Strategy, which was adopted in November 2020. A particular focus of the dialogue will be the development of production capacity for critical APIs, raw materials, and medicines in the European Union to ensure better preparedness for future pandemics, and will serve to put forward a set of policy recommendations to address the identified vulnerabilities by the end of 2021.

The launch event was opened by Margaritis Schinas, EC vice-president, with Stella Kyriakides, EC commissioner for Health and Food Safety, and Thierry Breton, EC commissioner, Internal Market, acting as moderators of the roundtable discussion with stakeholders. The Portuguese Presidency was represented by Marta Temido, the country’s minister for Health, and the European Parliament was represented by Dolors Montserrat and Nathalie Colin-Oesterlé—both ministers of the European Parliament.

The EC has launched a structured dialogue aimed at gaining a better understanding of global medicines supply chains and to identify potential causes and drivers of vulnerabilities.

EC Launches Structured Dialogue to Address Medicines Supply Vulnerabilities

A recent survey by SwedenBIO—a national non-profit association—has demonstrated a major upswing in the Swedish life science industry. The results of the survey were revealed in a March 2, 2021 press release.

The survey targeted pharmaceutical development companies with operations in Sweden and found that a vast majority (80%) of those surveyed are planning on expanding with new hires in the coming year. Additionally, when comparing with survey results from a year ago, it was revealed that there was increased confidence in projecting future growth with 62% of those surveyed expressing a more positive view of projections now.

“The positive outcome of our follow-up survey is very encouraging for the future,” said Helena Strigård, director general, SwedenBIO, in the press release. “The results also confirm our belief that involvement with other global life science hot spots opens more opportunities for collaboration and drives growth. We have, in fact had several fruitful interactions with similar organizations in regions internationally, including California. Furthermore, the fact that overall, the disturbances due to COVID have been limited and that numerous Swedish companies have re-focused their development towards COVID-related tests or treatments, underscores the strength of the industry as a whole and how creative and dedicated our life science industry experts are.”

Further information about the survey and a special investment conference being held in April 2021 can be found on SwedenBIO’s website.

A recent survey by SwedenBIO has demonstrated a major upswing in the Swedish life science industry. 

Survey Demonstrates Upswing in Swedish Life Sciences Growth

Merck (known as MSD outside the United States and Canada) will receive up to $268 million from the Biomedical Advanced Research and Development Authority (BARDA), US Department of Health and Human Services, to adapt and make available some of the company’s manufacturing facilities for the production of COVID-19 vaccines and medicines.

On March 2, 2021, the company announced multiple agreements with BARDA and Janssen Pharmaceuticals, Inc., one of the Janssen Pharmaceutical Companies of Johnson & Johnson (J&J), to support the manufacturing and supply of J&J’s single-dose COVID-19 vaccine. Merck will produce drug substance, formulate, and fill vials of the vaccine at US facilities.

Biden Administration invoked the Defense Production Act 

to equip the Merck facilities for this “historic manufacturing partnership” of two pharmaceutical giants, a White House spokesperson said.

“We are steadfast in our commitment to contribute to the global response to the pandemic as part of the remarkable efforts of the entire medical and scientific community,” said Mike Nally, executive vice president, Human Health at Merck, in the press statement. “This funding from BARDA will allow us to accelerate our efforts to scale up our manufacturing capacity to enable timely delivery of much needed medicines and vaccines for the pandemic.”

“At Merck, we have a rich legacy in vaccine manufacturing and look forward to combining our expertise with Johnson & Johnson to help increase supply and expand access to authorized SARS-CoV-2/COVID-19 vaccines,” said Sanat Chattopadhyay, executive vice president and president, Merck Manufacturing Division, in the statement.

anssen COVID-19 vaccine received Emergency Use Authorization from FDA on Feb. 27, 2021 

and initial supplies of vaccines shipped on March 1, 2021.

Merck discontinued development of two COVID-19 vaccine candidates

, when studies showed the immune response was inferior to levels following natural infection. The company announced plans to focus research and production capabilities on two COVID-19 therapeutic candidates.

With funding from the federal government, Merck will expand capacity to produce J&J’s COVID-19 vaccine.

Merck to Manufacture J&J Vaccine

AstraZeneca has announced, in a March 2, 2021 press release, that the mass rollout of its COVID-19 vaccine has begun via the COVAX initiative. These first steps by the company in the distribution of the vaccine to low- and middle-income countries will fulfill its efforts to provide broad and equitable access.

So far, shipments of the vaccine have been dispatched to Ghana and Cote D’Ivoire with more expected to begin arriving in the Philippines, Indonesia, Fiji, Mongolia, and Moldova shortly. Vaccine shipment allocations have been made according to the COVAX Allocation Framework, which determines volume per participating country based on several factors, such as readiness and national vaccination plans.

“These first steps towards fulfilling our broad, equitable, and no-profit emergency response to the pandemic mean millions of people, irrespective of their country’s income level, will soon be protected against this deadly virus,” said Pascal Soriot, CEO, AstraZeneca, in the press release. “This is a moment of great pride for us at AstraZeneca and I am extremely grateful to our partners including Gavi, CEPI, and Oxford University for their hard work and dedication in order to make this humanitarian ideal a reality for many millions of people around the world.”

“Global, equitable access to COVID-19 vaccines is only possible when the public and private sectors work together,” added Seth Berkley, CEO, Gavi, in the press release. “When we launched the Gavi COVAX Advance Market Commitment in June 2020, our first Agreement was with AstraZeneca. Nine months later, the first doses are already being delivered to those that need them most. This is the beginning of COVAX’s effort to end the acute phase of the pandemic, not the end, but we can all take strength from this moment and I thank AstraZeneca and the University of Oxford for their support and partnership at every step of our journey.”

AstraZeneca has announced that the mass rollout of its COVID-19 vaccine has begun via the COVAX initiative. 

AstraZeneca Progresses Rollout of COVID-19 Vaccine to Low- and Middle-Income Countries

WuXi AppTec announced on March 2, 2021 that it has acquired OXGENE, a UK-based contract research and development organization focused on the design and development of scalable gene therapy technologies.

Under the terms of the acquisition, OXGENE will become a fully owned subsidiary of WuXi Advanced Therapies (WuXi ATU), WuXi AppTec’s cell and gene therapy contract testing, development, and manufacturing organization (CTDMO) business unit, WuXi Apptec said in a company press release. OXGENE will be WuXi ATU’s first facility in Europe and will work to enhance WuXi ATU’s abilities to transform the development, testing, manufacturing, and commercialization of cell and gene therapy products while accelerating their time to market.

“OXGENE is proud to have invented a range of next-generation platforms that address the complexities of viral vector manufacturing for gene and cell therapies,” said Dr. Ryan Cawood, founder and CEO of OXGENE, in the press release. “We are excited to join the team at WuXi ATU and to work together to scale up our technologies as a combined business, which will strengthen and broaden our service offerings for customers, improving by orders of magnitude the scale and cost of bringing cell and gene therapies to market. The benefits will be significant for the industry and for patients worldwide.”

“We are delighted to welcome OXGENE to WuXi ATU,” added Dr. David Chang, CEO of WuXi ATU, in the press release. “By combining WuXi ATU’s world-class cell and gene therapy CTDMO platform with OXGENE’s innovative capabilities, we will be able to provide transformative solutions for our customers. This business combination represents a significant step in our ongoing effort to enable our customers and partners worldwide to deliver more effective and accessible advanced therapies to patients globally.”

Under the terms of the acquisition, OXGENE will become a fully owned subsidiary of WuXi Advanced Therapies, WuXi AppTec’s cell and gene therapy contract testing, development, and manufacturing organization business unit.

WuXi AppTec Acquires OXGENE to Increase Cell and Gene Therapy Offerings

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.

For a vaccine to be effective, the components of the disease of interest that provide protection from disease (an antigen) must be incorporated or present in the candidate and induce a strong and appropriate immune response in the vaccine. Viral vaccines specifically can be classified into ‘classic or traditional vaccines’ and include attenuated/killed virus. Recombinant vaccines include subunit, virus-like particle (VLP), conjugate, and viral-vectored vaccines. Genetic vaccines include messenger RNA (mRNA)/DNA. Alternatively, vaccines can be classified into live and non-live vaccines, which is important when considering safety profiles.

Traditional vaccines and viral subunit/proteins directly elicit an immune response without the need for cellular transcription and/or translation of genetic material. Genetic vaccines use part of the virus’ own genetic code (either mRNA or DNA) to instruct cells to make a viral antigen that will stimulate an immune response. Viral-vectored vaccines allow selection of a non-disease causing, and in some cases, a replication-deficient virus to be used to carry and deliver the antigen of interest to the vaccine and offer a mixture of advantages and disadvantages of live and non-live vaccines.

Traditional vaccines have a long track record of safety and efficacy. They have been shown, according to Charles Christy, head of commercial solutions for the Ibex Dedicate at Lonza, to effectively treat global diseases and eradicate disease burden, or even eliminate diseases (polio, smallpox).

The immune response is triggered immediately with this type of vaccination; however, the body also has to fight the full viral or antigen load, which may facilitate adverse effects (e.g., feeling under the weather following the flu vaccine), notes Thomas Becker, site quality director for Recipharm.

The potential exists for adverse effects because live viral vaccines are attenuated by genetic mutation of the wild-type, disease-causing virus, either by passaging the virus through cells, eggs, or animals or purposeful deletion of sections of the viral genome, explains Kelly Lyn Warfield, vice president of vaccines research and development within Emergent BioSolutions’ Vaccines Business Unit. “For selection and use of live, attenuated viral vaccines, caution must be applied due to potential safety issues in immunocompromised individuals (i.e., primary immunodeficiencies, patients on immunosuppressant treatment, HIV-infected people, and sometimes the very young or old), since this type of vaccine has the potential to replicate in an uncontrolled manner, spread to other individuals due to shedding of the vaccine, or revert to a virulent (disease-causing) form,” she says. “Care must be taken, therefore, to select a candidate that has an appropriately balanced replication profile to maintain an acceptable safety profile but that can also induce a potent immune response,” Warfield adds.

To modulate the immune response and to increase the duration of the immunization, Becker says that the entire vaccination program may have to be applied in several doses, which can be inconvenient for patients. Live viral vaccines may also be challenged with stability issues and require frozen temperatures to remain viable and potent.

Similarly, a viral vector-based vaccine can cause an immune response to the viral vector itself in addition to the antigen of interest for which it is delivering the nucleic acid, according to Gregory Bleck, vice president of research and development at Catalent Biologics. “This issue can make repeated dosing difficult, since with additional dosing, the patient’s immune system may clear the viral vector before it has an opportunity to insert the nucleic acid into a cell for antigen production,” he observes.

There are a multitude of approaches for non-live viral vaccines that can be based on whole, killed organisms; purified or recombinant proteins; VLPs; or genetic vaccines, notes Warfield. “In contrast to live vaccines,” she comments, “non-live vaccines pose no risk to immunocompromised individuals and cannot spread or potentially cause disease.” Development of purified or recombinant proteins, VLPs, and genetic vaccines, however, requires knowledge of the protective antigens, including their identities and possibly structures, to ensure proper and robust immune responses, she adds. In addition, some protein-, VLP-, and killed virus-based non-live vaccines require addition of an adjuvant to improve their immunogenicity due to their non-replicating nature.

Genetic vaccines are different because they contain only a small part of the virus’ genome and cause cells to produce the desired antigen(s). Typically, the RNA or DNA sequence that instructs the human cell to produce the viral antigen(s) intended to trigger an immune response is formulated in a lipid nanoparticle that can enter the human cell after vaccination, according to Becker. He adds that because the human cell produces the antigen itself in order to produce the immune response, a smaller dose of vaccinating agent is required for genetic vaccines when compared with traditional vaccines.

Some people consider viral-vector vaccines, in addition to those based on DNA and RNA, to also be genetic vaccines, because the genome fragment is embedded within a virus that usually cannot multiply itself in the human body, but once inside the cell directs the cell to produce the viral antigen.

In the case of DNA-based vaccines—either plasmid/naked DNA- or viral-vector-based—explains Bleck, the cell that takes up the DNA will transcribe the DNA into mRNA, and then the mRNA will be translated into the protein-of-interest, which will be an antigen from the virus. In the case of mRNA vaccines, the transcription step is already completed, so the cells that take up the mRNA only need to transcribe the mRNA sequence into the protein-of-interest. Viral vector-based vaccines utilize the engineered virus to insert the nucleic acid encoding the viral antigen into the cell, where that molecule is then subsequently transcribed to mRNA and translated into the protein-of-interest.

DNA- and RNA-based genetic vaccines have been impeded by the ability to appropriately deliver the nucleic acids intact and to the correct site within the body or a particular cell type, according to Warfield, but this issue has more recently been overcome with the development of novel, improved delivery formulations such as more effective lipid nanoparticles.

Importantly, although they are considered genetic vaccines, the DNA of the human cell is not impacted by the vaccine, and Becker notes that antigen production only continues for a short duration before the RNA or viral vector are eliminated from the body. As a consequence, though, the immune reaction (and potential adverse effects) occurs in a more modulated and delayed manner, Becker asserts. “As a result, to achieve complete immune protection and to increase its duration, a prime boost dosing regimen in two consecutive vaccinations with the same or a different vaccine may be applied, which is what we’re seeing in the mRNA COVID-19 vaccines,” he says.

Indeed, while genetic vaccines hold much promise, they have not yet been widely commercialized. This situation has started to change with FDA granting Emergency Use Authorizations to the mRNA vaccines from Pfizer-BioNTech and Moderna for COVID-19 in late 2020 and approval for the viral-vector vaccine for Ebola from Janssen (Ad26.ZEBOV) followed by a second dose of MVA-BN-Filo from Bavarian Nordic, also in 2020.

Development and manufacturing advantages for genetic vaccines

What makes genetic vaccines so attractive is the potential to establish platform solutions that allow the rapid development of a range of vaccines against emerging infectious diseases based on a scalable novel core technology platform. “The process to develop and produce genetic vaccines is highly similar from one to the next, so we have already a lot of information from other vaccine projects that have used the same platform. This saves valuable time and energy when starting the development of a vaccine against a new disease,” explains a company spokesperson from Janssen Infectious Diseases & Vaccines.

The design and development of traditional vaccines is fairly complex. Most importantly, the best way to propagate the virus or its antigen needs to be newly developed in each and every case, Becker stresses. For killed or attenuated viruses, a mechanism must be explored and established that kills or lowers the infectious potential of the virus, without destroying it, so that the ability to generate the immune response remains. If this is not achievable, the traditional vaccine route is blocked for these viruses. For conjugated or VLP vaccines, the appropriate carrier substance or partner molecules also need to be explored and then combined with the antigen in the formulation process.

“While historical knowledge can be applied in the attenuation or formulation process, each case requires developmental activities to some extent. The more similar the new virus is to existing organisms, the easier it will be to use existing processes, reducing the amount of development time required. This is particularly true when creating vaccines for new mutations of known and treatable viruses such as flu,” Becker remarks.

“Traditional vaccines can take years to develop because they rely on the actual virus or viral proteins grown within eggs or cells,” Bleck agrees. “Viral proteins are developed and manufactured in cell-based expression systems, and they require time to generate a clonal cell line expressing the protein-of-interest and then optimize the manufacturing process so that it can be reliably scaled-up to clinical and commercial scale.” Traditional vaccines based on cell culture are also manufactured using large stainless-steel equipment, which is slower to scale up and requires longer construction timelines, Christy adds.

In addition, traditional vaccines are usually ranked as requiring an elevated biosafety level, and manufacturing facilities therefore need to comply with a higher level of biosafety requirements. “Such capacities may be limited worldwide, especially in pandemic situations, and facilities that are usually used for the manufacturing of ‘normal’ drug products cannot be used. Moreover, the virus propagation or the production of the antigens are biochemical processes that need to be run under controlled conditions and that may take a long time (weeks to months),” Becker observes.

Genetic vaccines, on the other hand, can be designed more rapidly and deployed as soon as the genetic sequence has been identified. The DNA product can be quickly expressed and scaled-up to clinical or commercial scale, Bleck adds. In the case of mRNA vaccines, the DNA precursor can then be transcribed in an 

 process that does not require the same long development timelines; for viral vector-based vaccines, the base systems are already established and the gene that encodes the antigen of interest only needs to be substituted into the vector backbones to start the process. Subsequent engineered viral production is performed using well-established manufacturing techniques.

As an example, Christy notes that Moderna went from sequence selection to shipping the first manufactured batch of the clinical drug product in 42 days. In addition, Lonza was able to scale up, build, and commercialize the Moderna vaccine in its Visp, Switzerland facility in only eight months, compared to the two to three years (or more) usually required to build a new cell-culture facility. This speed was also made possible by Lonza’s pre-investment Ibex Dedicate in a manufacturing complex and supporting infrastructure that was pre-primed and ready for fit-out, according to Christy.

“Part of the reason for this speed is that no cell bank or viral seed bank needs to be developed; this time-consuming task is often required for traditional vaccines,” says Christy. “Secondly,” he continues, “the dose for genetic vaccines is very low compared to traditional vaccines; an mRNA dose of 30–100 µg is being utilized. Such low doses require smaller facilities, which can be equipped with single-use (SU) technology, resulting in a faster scale-up and facility implementation.” Similarly, viral vaccines can be produced in small SU bioreactors, with 1000-L vessels sufficient for producing millions of doses.

“Theoretically,” adds Richard W. Welch, vice president of development services for Emergent BioSolutions’ CDMO Business Unit, “once a process and testing have been developed for a genetic or platform-based vaccine, that same process and the majority of the release assays can be used for any vector or antigen. For more traditional vaccines, each process and set of release assays must be developed separately.”

More specifically, the level of development effort required for platform-based vaccines is often substantially less than for more traditional vaccines from a chemistry, manufacturing, and controls perspective, Welch explains. “This decreased level of process and method development results in a substantial reduction in cost, and more importantly during a pandemic, the time from determination of identity and sequence of the target antigen or antigens sequence to clinical production and release of clinical trial material.”

Moreover, the biosafety level category for genetic vaccines usually is lower than for traditional vaccines. “The majority of these vaccines may not even require biosafety measures during production at all, merely the same techniques as those used for other biopharmaceutical products,” notes Becker. As such, facilities that produce “normal” biopharmaceutical drug products can be used also for the manufacturing of these vaccines.

In addition, Welch observes that a genetic- or platform-based approach can also avoid other potential delays for clinical production. “If a site has already manufactured, tested, and released that type of genetic or platform vaccine, then the equipment is in place and qualified for any vaccine that platform can support. Materials and suppliers are already qualified, and materials specifications are already developed and set. Often, long lead-time items with long expiry dates can also be held in stock, decreasing the time necessary to order, release, and supply material to the manufacturing or QC [quality control] suites,” he says.

However, Becker points out, genetic vaccines are a much younger class of drug product compared with traditional vaccine techniques. “The knowledge level for some of the required production techniques, such as the formulation of lipid nanoparticles, is still limited within the pharmaceutical industry. We can expect to accrue even more valuable knowledge over time to further enhance the effectiveness and efficiency of these vaccines,” he states.

Easy to modify in response to virus mutations

Viruses naturally mutate, and the longer they are in circulation and the more transmission that occurs, the more opportunities they have to develop mutations that enable resistance to established antigens. Depending on the mutations, the impact can range from minimal to substantial for traditional vaccines, according to Welch. Traditional flu vaccines offer an existing case where the seasonal variation has minimal to no impact on the production process. However, substantial mutations or changes in strains with increased potential for infection can result in substantial changes.

“If the mutation or shift in strains cannot be produced using the existing process, such as an avian flu not being able to be produced in eggs, then inactivated and attenuated vaccines could potentially require completely new starting strains or production platforms, which would essentially require anything from substantial to complete redevelopment and validation of the manufacturing process for the new strains,” he says. The same could hold true for protein-based or recombinant protein-based.

Because genetic vaccines trigger their immune response via a fragment of the virus genome, they will continue to provide an effective immunization route as long as the gene sequences in those particular fragments remain unchanged or display minimal mutations, according to Becker.

If the mutation is more significant, genetic vaccines also have the advantage of being extremely easy to modify. “That is the beauty of genetic vaccines; they represent a platform approach with a central technology (adenovirus, liposome particle mRNA, or other) that can be readily modified by inserting a new genetic sequence for rapidly response to virus mutations or even novel viruses (e.g., other coronaviruses),” Christy asserts.

The DNA template just needs to be modified to encode the new variants, and manufacturing of the updated vaccine can typically continue with very few, or no modifications to the process, Bleck notes. The existing body of knowledge about related virus strains facilitates the location and isolation of the relevant gene sequence, Becker says. Bleck adds, however, that the new vaccines still need to be evaluated in detailed clinical studies to confirm that the new mutated epitopes do not elicit unwanted immune responses in patients.

Like with traditional vaccines, developers of genetic vaccines must ensure sufficient process and product understanding to meet FDA, European Medicines Agency (EMA), and other regulatory guidelines, and they face scalability issues moving from early clinical phase to late clinical phase and commercial. “One key difference for genetic vaccines,” asserts Welch, “is that if the technology is a true platform approach, these issues only have to be worked out once for multiple indications.”

Companies, however, still need to define how much volume should be manufactured in order to meet demand and ensure that they—or their manufacturing partners—have the capacity to scale-up operations to meet the necessary peak volume. Genetic vaccines, while faster to develop than proteins, are still biologic products that require cell culture and sophisticated technology for development and manufacturing at both clinical and commercial scale, according to Bleck.

In a pandemic, adds Christy, the key challenge is manufacturing at risk during ongoing clinical trials. Further, the production scale-up to deliver hundreds of millions (if not billions) of doses places an enormous strain on the supply chain. Becker points out that currently there is only limited global capacity for biopharma production, some of which is required for existing drug and vaccine products. In addition, both traditional and genetic vaccines require filling under aseptic conditions, further reducing global capacity.

Genetic vaccine approaches have been contemplated for nearly 30 years, but until recently, Warfield says, they had been challenged by the need for large doses to induce mild to moderate immune responses, the instability of the nucleic acids (particularly with RNA candidates), and the inability to efficiently deliver the nucleic acids to targeted tissues or cells.

The recent success of the Pfizer-BioNTech and Moderna mRNA vaccines demonstrates that these issues have been overcome at least to some degree, but Warfield believes further improvements will need to be made for continued and future success of this class of vaccines. She highlights a need for the reduction of costs for raw materials and manufacturing processes and improvements in stability to reduce the need for storage at low or extremely low temperatures, which can create supply-chain challenges.

“The lower stability of genetic vaccines is definitely the major challenge when it comes to widespread adoption of this new technique,” Becker agrees. Formulation of the lipid nanoparticles required to protection of mRNA vaccines from degradation can be challenging, however; the correct ratio of lipids, proteins and nucleic acids is needed to form the nanoparticle, in addition to any additional excipients, adjuvants, etc., according to Bleck.

In addition, genetic vaccines may only undergo a limited number of freeze-thaw cycles, and thus the bulk vaccine should be filled into vials immediately after the formulation process is completed. In addition, he notes that the filling process should be followed immediately by visual inspection and label and pack operations. “With this in mind, the size of a batch, which is processed in one run, may be limited in order to shorten the exposure of the vaccine to room temperature,” he comments.

Because genetic vaccines are so new, there is also a need for careful short- and long-term monitoring and understanding of side effects to assist in the development of next-generation candidates that are potentially safer with less untoward effects, Warfield adds. “Achieving understanding and approval of the safety and efficacy profile of genetic vaccines by the general public, and especially amongst those with existing vaccine hesitancy, will be required for efficient and broad acceptance of genetic vaccines in the future for a larger set of pathogens outside the current COVID-19 pandemic,” she continues.

Overall, Christy observes that as with any new technology, genetic vaccines will progress through a maturation curve. Equipment and production processes must be optimized and standardized so processes can be made robust and scalable and global supply can be achieved at acceptable costs. He also stresses that the human capital factor must not be neglected. “The availability of trained, regulatory-compliant GMP manufacturing staff and quality control and quality assurance personnel is a critical success factor,” he says. Much investment in facilities will be needed as well, which during the COVID-19 pandemic been supported by numerous government and private partnerships.

A similar challenge common to all vaccines is the delivery of a safe and efficacious dose to patients. “Full focus on SISPQ (safety, identity, strength, purity, and potency) is common to all medicines. However, for genetic vaccines, this means developing a whole new battery of analytical methods, especially around purity and potency,” Christy states. “Methods exist, but these generally are research-based techniques (such as sequencing, infectivity, transfection assays), and they need to be fully developed to be robust, validatable, GMP-compliant, and able to be routinely implemented across multiple sites (globally for both drug substance and drug product),” he says.

Methods for genetic sequencing and product release, adds Bleck, require specialized expertise and equipment not required for traditional vaccines. In addition, he notes that for mRNA-based vaccines, their instability relative to most proteins will typically expand the scope of stability studies required.

The shortened timelines for manufacturing of genetic vaccines, Becker comments, also means that with currently accepted analytical methods, analysis results for intermediate production materials, such as formulated bulk product or product in filled but unlabeled vials, are not available at the time of processing. “This situation results in an increased risk that non-conforming product may undergo further value-adding manufacturing steps before being identified and discarded,” he says.

Because genetic vaccines are still a relatively new class of treatments, there is significant potential for further development. Primary opportunities revolve around the selection of more appropriate excipients, observes Welch. Work is ongoing to improve the target product profiles of genetic vaccines to improve their stability, Christy adds. “Formulation development with a range of stabilizers offers the potential to improve both the temperature stability and the shelf life of such vaccines,” he states.

In some cases, Welch believes that the use of existing or novel drying technologies will improve the stability of genetic vaccines. Becker agrees that transferring liquid formulations into lyophilized products can potentially improve thermal stability and reduce the need for cold-chain handling. Recipharm’s site in Wasserburg, Germany, for instance, successfully developed and implemented a lyophilization cycle that can freeze-dry a COVID-19 mRNA vaccine. “Initial results indicate that the lyophilized product has a significantly improved thermal stability profile in comparison to the liquid formulation,” Becker says.

The need for formulation improvement, however, creates its own challenges for vaccines being used to treat emerging infectious diseases, especially in the case of pandemics, where speed is of the essence, according to Welch. “Opportunities to increase stability that involve substantial revision of the final formulation may require additional and extensive clinical trials to establish efficacy and safety along with revalidation of the drug product production process.”

While there are many advantages to genetic vaccines, there are also many reasons why traditional vaccine approaches will continue to be used. “Traditional vaccines have been on the market much longer than genetic vaccines and, in most cases, can currently be made at a significantly lower cost,” Bleck. “Over time,” he adds, “these technologies have evolved to adapt to changes in the viruses that they protect against. In addition, if necessary, multiple antigenic proteins in certain ratios and configurations can be expressed to get the correct epitopes produced for a neutralizing immune response, which is currently difficult to achieve in genetic vaccine formats. Furthermore, getting adjuvants to work effectively with genetic vaccines can also be difficult.”

Traditional approaches might be preferential when preclinical animal studies indicate that an effective vaccine requires the presentation of an antigen or antigens in a complex structure that cannot be engineered or can only be effectively and reproducibly done using the entire infectious agent, a company spokesperson from Janssen Infectious Diseases & Vaccines says. In addition, when the protective antigens are not known for an emerging pathogen, traditional approaches incorporating the entire pathogen (live or killed viral vaccines) may be required until a firm understanding of protective immune responses are established, Warfield notes.

Furthermore, Becker points out that traditional approaches allow the combination of vaccination agents for different diseases, which may not be possible for genetic vaccines for the foreseeable future, as they are less robust. “With this in mind,” he says, “traditional vaccines may be more appropriate not only to address a wider variety of mutations of one virus type in one vaccination, but for routine vaccination programs against different diseases.”

Finally, Warfield notes that killed virus, protein, and VLP vaccines potentially have the advantage of more temperate storage requirements compared to some live virus- and genetic-based vaccines, which can be particularly important in remote areas and developing countries that lack the necessary cold-chain infrastructure.

But genetic vaccines are creating real excitement

The arrival of the new class of genetic vaccines heralds an exciting time in vaccine development, asserts Becker. “This new technology offers a number of promising routes to help us not only enhance infectious disease prevention, but to progress the fight against other serious diseases, such as cancer,” he says. The fact these vaccines can be manufactured in standard multi-product facilities, rather than on specialist sites, will also enhance efficiency for pharmaceutical companies and offers greater scope for maximizing vaccine production capacity in the event of future pandemics.

Process and product regulations for genetic vaccines and viral-vectored vaccines still have to mature, however, as the technologies thus far have only recently produced commercially viable products that have been approved by stringent regulatory agencies like FDA and EMA. “Our expectation is that both technology regulations and platform regulations will evolve over the next several years,” says a company spokesperson from Janssen Infectious Diseases & Vaccines.

As more genetic vaccines become available, Welch envisions a shift to their use not only for pandemics, but also for existing and emerging infectious diseases. “Such as shift could enable companies to decrease time and cost, at least for manufacturing development, from inception to market,” he notes.

New advances in mRNA vaccines and other novel vaccines may indeed mean that we are entering into a new “golden age” for vaccines, according to Christy, but with the caveat that important work remains to be done to ensure global access, adequate capacity, and ease of administration, as well as education to ensure widespread uptake. He also notes that effective vaccines have yet to be developed for many well-known viruses, and more than 80 emerging viruses have been identified since 1980, with two novel viruses appearing every year. “Despite recent successes, much remains to be achieved to protect humanity,” he insists.

In addition to novel genetic vaccine technologies, Christy is excited about mucosal vaccines that aim to boost local immune memory and effector responses at the point of entry of pathogens and mRNA vaccines in development as cancer vaccines, intra-tumoral immune-oncology therapies, and localized regenerative therapies. Other cancer and T-cell vaccine are in development to treat antibody evasive viruses, and existing vaccines are being re-developed to provide better protection and coverage.

“Ongoing global pre-investment by governments, private foundations, and other groups will be needed to drive further advances in technology, manufacturing capacity, and infrastructure that will enable effective response to epidemics/pandemics going forward,” Christy concludes.

Cynthia A. Challener, PhD, is a contributing editor to 


Vol. 34, No. 3
March 2021
Pages: 10–17

When citing this article, please refer to it as C. Challener, "Genetic Vaccine Platforms Demonstrate Their Potential, "

In a pandemic, genetic vaccines offer several advantages over traditional approaches.

Genetic Vaccine Platforms Demonstrate Their Potential

Feliza Mirasol is the science editor for 

The use of seed trains to increase product yield is common to cell culture processes; however, the process can be costly and time-consuming. Seed train intensification technologies have been useful, however, in achieving the desired cell density for inoculation into a bioreactor while shortening the seed train duration, which can optimize biological product output.

“The seed train is the scaling of the culture from a small volume of cells in a cell bank vial to a larger volume of cells that are used to inoculate the N, or main production reactor at the required cell density,” explains Christine Gebski, senior vice-president, Filtration and Chromatography, Repligen. The seed train process aims to ensure cell viability and the attainment of the desired or required final cell density.

“The purpose of the seed train is to reach a sufficient cell mass to inoculate the production bioreactor in order to start the protein production in an optimal way,” agrees Darren Verlenden, head of BioProcessing at MilliporeSigma. Verlenden notes that, from the working cell bank vial to the production bioreactor, the cell number has to increase approximately 6–8 logs. “This [increase] is typically achieved by running cells through many cultivation systems (e.g., spin tubes, shake flasks, rocking motion bioreactors, stirred tank bioreactors), which become larger with each passage.”

While each process is different, typical seed trains are time consuming and labor intensive and have relatively low value-add, Verlenden says. In addition, the risk of failure remains significant because of the open nature of the early stages of cell expansion when working cell bank vials are thawed and cells are manually transferred into spin tubes and shake flasks under a laminar-flow hood.

Incorporating seed train intensification can benefit the overall manufacturing strategy. As Verlenden explains it, seed train intensification aims to lower manufacturing costs, achieve higher process throughput, and increase flexibility.

Gebski adds that increasing the cell density of the cell bank or the N-1 reactor enables a shorter timeframe to attain the higher cell densities needed in the “N” or main production bioreactor. “Reducing process time drives meaningful efficiency gains, with a larger number of processes being completed in a given amount of time. Attainment of a higher cell density directly correlates to an increase in productivity—the amount of biologic that can be produced in a set amount of time.”

Two main options are currently being assessed or are already implemented in the biopharmaceutical industry to intensify seed trains, says Verlenden. These options are based on perfusion: N-1 perfusion and high cell density cryopreservation (HCDC). Verlenden explains that perfusion operation is based on the introduction of fresh cell-culture media at a constant rate in the bioreactor, while the spent media is removed and cells are returned to the bioreactor vessel using a cell retention device. “This way the nutrients and waste-product content remain stable over time, which allows greater cell mass levels in the bioreactor,” he says.

Improved and simplified process controls and cell retention technologies have allowed greater adoption of perfusion, he notes. Verlenden also notes that drug manufacturers are assessing the implementation of perfusion in the N-1 bioreactor to reach high cell densities. “N-1 perfusion can be used in two different ways. The first and the most impactful is to use N-1 perfusion in order to seed the production bioreactor with higher initial cell density, known as high seed fed-batch. The other way to use N-1 perfusion is to take advantage of the higher cell density to use a smaller bioreactor and seed the production bioreactor,” he says. In both cases, the advantage of using N-1 perfusion is to keep the production bioreactor operation in fed-batch mode, which inherently eases the process validation, especially post-approval changes, Verlenden says.

Meanwhile, to reduce the duration of the cell expansion period, drug manufacturers have developed higher-density cell banking approaches in larger vials (5 mL), resulting in some improvements but without alleviating the contamination risk due to manual transfers of cells, Verlenden states. “This concept has evolved with the use of single-use 2D [two-dimensional] bags of 100 mL–250 mL volumes with high cell densities (100–150 x106 cells/mL) generated by perfusion processes in HCDC settings. From a 250-mL seeding single-use bag at 100 x106 cells/mL, you can directly spike a 20-L rocking motion bioreactor or a 50-L stirred-tank bioreactor to start the cell expansion—reducing the number of steps and accelerating speed,” he says.

Verlenden also points out another benefit of using HCDC bags, which is to make the whole seed-train functionally closed. “After thawing the bag, you can weld it to the bioreactor and transfer cells aseptically, allowing drug manufacturers to start the seed train in a less controlled area to save time on environmental testing,” he says.

Using perfusion in the seed train, whether in a N-1 perfusion scenario or HCDC for cell banking, is a first step into upstream intensification—from a process control and cell retention technology standpoint. Perfusion also provides opportunities for cost-of-goods improvements and inherently increases product safety, Verlenden adds.

With the use of seed train intensification in the manufacturing process, bioreactor design has been adapted to keep pace and innovative technologies have helped with that adaptation.

There are obvious challenges to enable high cell densities from a bioreactor perspective, says Verlenden. Sparging and mixing capabilities, for instance, are essential to achieving the right balance between maximizing the oxygen transfer rate and minimizing the shear stress imposed on the cells in suspension.

“Today, kLa [volumetric mass transfer coefficient for oxygen] up to 50/h and power input per volume up to 100 W/m3 at 2000-L scale are becoming the norm in single-use bioreactors. Bioreactor vessels geometry improvements, design optimization of impellers, and the diversification of drilled hole spargers have been instrumental in achieving these specifications,” Verlenden says.

Meanwhile, he continues, from a single-use bioreactor perspective, the emergence of new films for 3D bags that are more resistant to mechanical constrains are alleviating the roadblock to running a single-use bioreactor in perfusion for extended duration. And from a seed train intensification perspective, perfusion is being made easier with new technology that is based on tangential flow filtration (TFF). This new TFF-based technology (Cellicon Solution, MilliporeSigma) is specially designed for seed train intensification. “Coupled with modern bioreactor designs, this is currently making perfusion more attractive, especially for N-1 perfusion and HCDC cell bank manufacturing,” says Verlenden.

Proven and scalable cell retention devices (e.g., XCell ATF Technology, Repligen) have enabled N-1 cell culture intensification processes up to 3000 L to seed 15,000-L to 18,000-L reactors globally, adds Gebski. Along with bioreactor design and control systems, these devices have enabled the higher cell densities attained with intensified seed train culture, she says.

Are there particular synergies to using seed train intensification together with single-use bioreactors and/or single-use technologies, or is the end result (optimized yield) the same whether using single-use bioreactors or stainless-steel? Verlenden says that the single-use vs. stainless-steel factor depends on the manufacturing context. “Some companies have made the choice to implement N-1 perfusion in stainless-steel bioreactors with volumes greater than 3000-L to seed 10-L to 15,000-L stainless-steel bioreactors operating in typical fed-batch. This allows maximum productivity in fed-batch at large scale for commercial molecules while taking advantage of seed train intensification,” he says.

Gebski also says that the seed train intensification process and outcome is the same whether stainless-steel reactors or single-use reactors are used. “Seed train intensification, and the benefits provided, continue to be demonstrated and clinically or commercially implemented in many mid-scale and large-scale cell culture processes,” she says.

In addition, she makes the distinction that single-use components and technologies are implemented for their shorter implementation time, reduced equipment cleaning requirements, and ease-of-use. “Single-use has not paved the way for seed train intensification approaches, but rather single-use and intensified seed train approaches drive efficiency gains into the manufacturing process, regardless of bioreactor type,” she states. The addition of closed, single use alternating tangential flow devices has simplified and expedited this adoption even further aligning with the trend towards single use facilities.

Meanwhile, as perfusion in the production stage with continuous harvest of proteins becomes more adopted in the industry, the concept of scaling processes out—as opposed to scaling processes up—will unleash its full power, Verlenden states. “This will be only possible with extensive use of single-use bioreactors and components in order to maximize process flexibility,” he adds.

The use of single-use technologies is a key enabler of process intensification overall, Verlenden also says, citing the Biophorum Operations Group’s technology roadmap issued in 2017 (1). He notes that the enabling benefit of single-use technologies is even more true in the seed train, where typical early stages are manual and open. The use of single-use technologies, such as HCDC bags, make the entire upstream process functionally closed, which ultimately reduces the burden and costs associated with environmental testing required for room classification, he explains.

“The adoption of intensified seed train at larger scale—late-stage clinical and commercial manufacturing—remains slower than initially anticipated by the industry, and this is not necessarily because of a lack or inefficiency of single-use technologies,” Verlenden goes on to clarify. He explains that the long-standing perception within the industry is that media cost for implementation of perfusion in the seed train outweighs the benefits of increased throughput and process flexibility. “While a greater quantity of media is needed to run a N-1 bioreactor in perfusion, recent cost model data clearly show that the media price and cost have a [negligible] impact on the upstream manufacturing costs per grams of protein produced. A better understanding and use of process economics modelling would likely alleviate this industry perception in the future,” he concludes.


Vol. 34, No. 3
March 2021
Pages: 20–22, 30

When referring to this article, please cite it as F. Mirasol, “Jumping Seed Train Intensification Hurdles to Maximize Yield,” 

Overcoming time and cost constraints can help enable seed train intensification efforts to maximize product yield.

Jumping Seed Train Intensification Hurdles to Maximize Yield

Garima Thakur is a graduate student in the Department of Chemical Engineering at the Indian Institute of Technology New Delhi.

Anurag S. Rathore is a professor in the Department of Chemical Engineering at the Indian Institute of Technology Delhi and a member of 

l's Editorial Advisory Board, Tel. +91.9650770650, asrathore@biotechcmz.com.

Process analytical technology (PAT) is an increasingly important aspect of biopharmaceutical manufacturing processes. FDA in 2004 described a regulatory framework for PAT and has been urging the industry to voluntarily develop innovative tools and techniques for quality control and assurance in biopharmaceutical manufacturing unit operations (1). The quality-by-design (QbD) framework encourages the use of PAT tools to “build quality into the process” rather than “testing quality into the product” (2). The importance of PAT tools is amplified in the case of continuous manufacturing processes, where individual batches are not well-defined and critical quality attributes (CQAs) of the therapeutic must be consistently maintained, monitored, and controlled over months of operation, rather than simply tested at the end of each unit operation or each batch (3–4). The need for real-time monitoring and control of CQAs and critical process parameters (CPPs) in continuous manufacturing has led to the adaptation of traditional end-of-batch quality testing techniques, such as analytical high-performance liquid chromatography (HPLC) and ultraviolet-280 (UV-280) spectroscopy, into in-line at-line modalities with periodic sampling from continuous unit operations (5–7). It has also spurred development of new analytical tools that provide information in near real-time and can be placed in-line in process flow streams.

In-line spectroscopic sensors using infrared, near-infrared, or Raman spectroscopy are one such new class of tools finding multiple PAT applications in bioprocessing (8). Spectroscopic PAT applications typically consist of in-line sensors combined with statistical or modeling methodology to monitor, control, and/or predict biotechnology processes. The key advantage of spectroscopy is the ability to gain information about the presence and quantity of multiple analytes in the process mixture simultaneously in a few seconds or less, overcoming a major analytical bottleneck and facilitating real-time control decisions (9). Spectroscopic techniques are also non-degradative and provide a wealth of spectral information that can be used in tracking process trajectories and flagging deviations (10,11). Thus, spectroscopic probes and flow cells are well-suited to PAT applications, particularly in tandem with multivariate data analytics (MVDA) tools, which are critical for reducing the dimensionality of large spectral datasets and extracting statistically significant quantitative information (12).

 Summary of spectroscopy-based downstream process analytical technology (PAT) applications in recent literature.

summarizes the recent literature of the past five years on implementing spectroscopy-based analytical tools in downstream bioprocessing (13–29). The use of spectroscopic immersion probes as PAT tools in upstream microbial and mammalian bioreactors has been extensively reported in the recent literature for identification and quantification of proteins, by-products, and substrates (30–32). However, their use in downstream processing is a more recent development. Near infrared spectroscopy (NIRS), in particular, has been demonstrated to have a wide variety of potential applications in different downstream unit operations, including capture chromatography (24), protein PEGylation reactions (25), and tangential flow ultrafiltration (26). These three case studies demonstrate the versatility of NIRS as a PAT tool and showcase the common underlying framework of the different applications, namely the collection of real-time spectra followed by comparison with spectral calibration libraries using multivariate statistical techniques. NIRS is demonstrated to be a reliable tool for acquiring rapid quantitative information in a range of downstream bioprocesses, and a key enabler for real time control.

Case study 1: NIRS for controlled loading

Protein A capture chromatography is typically the first downstream step in manufacturing of monoclonal antibodies (mAbs). One of the key difficulties in continuous Protein A chromatography is handling variability in the titer of the upstream material. The product titer is expected to change over time in the case of upstream perfusion cell culture systems (33). Even in the case of fed-batch processes, batch-to-batch variability necessitates flexibility in the downstream capture step (34). An increase or decrease in the protein concentration in the load stream affects the dynamic binding capacity of the Protein A resin, leading to changes in process performance (35). Over-loading the Protein A column with high-titer feed can lead to the loss of expensive mAb product, while under-loading decreases the resin utilization of the Protein A resin, one of the most expensive consumables that has been reported to contribute to up to 60% of the costs of the downstream processing train (36).

CLICK FIGURE TO ENLARGE. All figures are courtesy of the authors. 

 Near infrared spectroscopy (NIRS) as a process analytical technology tool for control of loading in continuous Protein A chromatography by monitoring load titer and column breakthrough percentage in real time. MVDA is multivariate data analytics.

has been used to address this challenge of handling potential titer variability in continuous capture chromatography while maintaining resin utilization and preventing both under- and over-loading (24). NIRS flowthrough cells placed in the loading stream and the outlet of the load column were used to collect spectra of the harvest and column flowthrough every three seconds. These spectra were passed to online MVDA models calibrated with reference spectra to determine the concentration of mAb in the harvest and flowthrough to within ± 0.05 mg/mL. The real-time concentration data were used to calculate both the total mg of mAb loaded on the column as well as the percentage breakthrough from the column, and these were used to make control decisions to start and pause the loading in a three-column periodic counter current Protein A process on a continuous chromatography system (Cadence BioSMB, Pall). The normal operating range of the control system allowed for concentration variations in the harvest between 3–8 g/L, achieving optimal resin utilization as well as process scheduling within this range.

The NIRS-based PAT control strategy was tested by inducing a range of linear and non-linear deviations in the load titer in real time. The deviations were designed to closely simulate those which would potentially occur during a perfusion process. The NIR flow cells provided the advantage of monitoring not only loading but also changes in the column binding capacity in real time, which is useful for providing early warning of resin degradation or column quality issues and facilitating optimal loading without relying on adsorption isotherm models, which may not be valid after multiple cleaning cycles. The system allowed resin utilization to be maximized, lowering consumable costs by ensuring that the total protein processed per mg of resin was consistently maintained. The NIRS flow cells enabled online measurement of concentration and facilitated real-time control decisions for increased efficiency, flexibility, and agility of the continuous chromatography process in the face of unexpected deviations.

Case study 2: NIRS for monitoring/control

Protein modification with biocompatible polymers, such as polyethylene glycol (PEG), is often used to improve the pharmacological properties and stability of biotherapeutics (37). Manufacturing of PEGylated drugs requires a PEG conjugation reaction to be carried out on the purified drug substance at the end of the downstream train (38). Proper control of the PEGylation reaction is critical to maximize PEGylation efficiency while minimizing the presence of over-PEGylated variants. Various critical process parameters affect PEGylation quality, including pH, reaction time, and the concentration and order of addition of the reactants (39). In the present case study, the PEGylation reaction of recombinant human granulocyte colony stimulating factor (rh-GCSF) was considered. The challenge was to monitor the PEGylation process trajectory and reaction kinetics to flag potential deviations in real time, as well as to control the reaction quenching to optimize the production of the desired monoPEGylated variant before its further conversion into undesired diPEGylated and multiPEGylated forms.

A few different PAT tools have been explored in the literature for monitoring and control of PEGylation. Some researchers used at-line size exclusion chromatography to monitor the progress of the PEGylation reaction, though this was not suited for real-time control due to the long method run times of >30 minutes for an overall reaction time of one to two hours (40). Other researchers used at-line matrix assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry for tracking the kinetics of PEGylation, though the time scale of analysis was again too long at over one hour (41). The use of NIRS as a PAT tool in the present case study overcame the bottleneck of analysis time and was able to track the conversion of rhGCSF into its monoPEGylated and multiPEGylated forms on a time scale of a few seconds (25). The NIRS spectra were acquired every three seconds and compared against a calibrated MVDA regression model, built using control runs of the reaction with orthogonal HPLC-based quantification of the PEGylated variants. The spectra acquired during the control runs were also used to develop a multivariate batch evolution model of the ideal reaction trajectory. A flow chart of the NIRS-based PAT tool is shown in 

 Near infrared spectroscopy (NIRS) as a process analytical technology tool in PEGylation reaction of recombinant human granulocyte colony stimulating factor (rhGCSF) by monitoring concentrations of mono- and di-PEGylated species over time and tracking deviations in the process trajectory.

The PAT tool was demonstrated in various control and deviated reaction runs in which online spectra were acquired by the NIRS immersion probe and used not only for quantification of the PEGylated variant, but also for comparison against the ideal reaction trajectory. This allowed process deviations to be identified and flagged in case a statistically significant difference was found between the trajectories of the current process versus the ideal one. A range of deviations were found to be identifiable by the PAT tool, including incorrect concentration of chemical additives, incorrect ratio of PEG to rhGCSF, incorrect order of addition of the reactants, and incorrect quenching time. The impact of each deviation on CQAs of the PEGylation reaction product was characterized, and each resulted in lower product purity and process yield. The NIRS probe was demonstrated to provide critical real-time information and form the basis of a robust PAT tool for monitoring and control of the PEGylation reaction of rhGCSF. The overall approach is generalizable to other downstream reaction steps, such as enzyme reactions or esterification.

Case study 3: NIRS for control of retentate concentration

Tangential flow ultrafiltration (UF) is a key unit operation in downstream processing of biotherapeutics, used for concentrating and buffer exchanging the in-process drug substance into the desired target concentration and formulation of the final drug product (42). This is typically the last unit operation prior to fill/finish and packaging. The concentration of the biotherapeutic in the final drug product is a CQA determined solely by this unit operation, necessitating robust control. In batch mode, concentration is achieved by recirculating the drug substance held in a large tank through the UF membrane until the desired volume reduction is achieved (43). However, batch-mode recirculation is not possible in the case of constantly incoming flow streams in continuous processing. The solution is to use single-pass UF in which the membrane module has a larger area and a long flow path, facilitating volume reduction of the feed stream in a single pass without the need for recirculation (44).

 Near infrared spectroscopy (NIRS) as a process analytical technology tool for control of retentate concentration in single pass tangential flow ultrafiltration in continuous formulation of monoclonal antibodies (mAbs). UF is ultrafiltration.

There are several operational challenges that must be overcome, however, to ensure that the retentate stream emerges from the single-pass tangential flow filtration (SPTFF) module consistently at the fixed target concentration. Reversible and irreversible membrane fouling as well as deviations in the concentration of the incoming flow stream from prior unit operations, such as polishing chromatography, lead to changes in the concentration factor achieved in a single pass (45,46). Therefore, a PAT strategy is needed to monitor the concentration of the incoming feed stream and control the flux across the membrane, to ensure that the retentate concentration does not vary over the course of the process. Researchers demonstrated a strategy leveraging in-line NIRS flow cells in the feed and retentate streams of an SPTFF module to make flux-based control decisions and ensure consistency in the retentate concentration, as shown in the schematic in 

The NIRS flow cells were able to measure the concentration of mAb in the range of 0.5–200 g/L, a significant improvement over UV-based quantification methods, using suitably calibrated spectral libraries. The control decisions were made on the basis of the real-time NIRS data as well as a pre-characterized design space for the limits of maximum flowrates and concentration factors achievable for a given feed concentration and flow rate within the pressure limits of the membrane module. The NIRS data were analyzed, and closed-loop control established with a permeate pump and retentate valve, allowing flux control on the time scale of <1 second. Control and scheduling decisions were made for integrating the SPTFF step with the rest of the continuous downstream train and for ensuring timely cleaning. The overall PAT strategy was demonstrated over two 12-hour case studies. The NIRS monitoring sensors enabled the development of a robust PAT control strategy to ensure that retentate concentration targets were consistently met over long continuous campaigns.

The need for rapid, non-degenerative, and robust analytical tools will continue to grow as the biotherapeutic industry embraces the PAT and QbD paradigms. Spectroscopic sensors are uniquely suited to fulfill this need as spectral information can be collected in a few seconds and analyzed in milliseconds using multivariate techniques. The spectra can yield a wealth of information about the in-process sample, including quantification of multiple analytes and the overall health of the process compared to an ideal trajectory. Spectroscopic applications in upstream microbial and mammalian cell cultures have grown exponentially in the past 10 years, and this can be expected to happen in the case of downstream unit operations in the near future. The rapidly growing interest in continuous downstream processes in both academia and industry will amplify this effect due to the unique real-time PAT control challenges that arise when batch-mode product quality testing is no longer an option. To truly bring spectroscopic applications into a manufacturing setting, however, the industry needs to gain confidence in the use of multivariate calibration models, which can only be done by having robust guidelines and frameworks for spectroscopic model calibration, validation, and periodic checking to account for shifts in spectroscopic sensor data over months or years. More fundamental studies are also needed to draw clear relationships between biotherapeutic CQAs and their effect on spectral features, as has been done in the case of generic pharmaceuticals. Finally, the ability of spectroscopic sensors to collect time-stamped spectral process signatures at high frequency over months or years is also a potential advantage that can be leveraged to create large-scale manufacturing datasets providing deep process history information, which can be of use to regulatory bodies.

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Integrated Continuous Biomanufacturing IV

The need for real-time monitoring and control has spurred the development of new analytical tools.

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